The term apoptosis first appeared in the biomedical literature to delineate a structurally distinctive mode of cell death. The cardinal morphological features are cell shrinkage, accompanied by bubbling and blebbing from the surface, and culminating in separation of the cell into a cluster of membrane-bounded bodies. Organellar structure is usually preserved intact, but the nucleus undergoes a characteristic condensation of chromatin, initiated at sublamellar foci and often extending to generate toroidal or caplike, densely heterochromatic regions. Changes in several cell surface molecules also ensure that, in tissues, apoptotic cells are immediately recognized and phagocytosed by their neighbors. The result is that many cells can be deleted from tissues in a relatively short time with little to show for it in conventional microscopic sections.
This process is responsible for cell death in development, normal tissue turnover, atrophy induced by endocrine and other stimuli, negative selection in the immune system, and a substantial proportion of T-cell killing. It also accounts for many cell deaths following exposure to cytotoxic compounds, hypoxia or viral infection. It is a major factor in the cell kinetics of tumors, both growing and regressing. Many cancer therapeutic agents exert their effects through initiation of apoptosis, and even the process of carcinogenesis itself seems sometimes to depend upon a selective, critical failure of apoptosis that permits the survival of cells after mutagenic DNA damage. Apoptosis probably contributes to many chronic degenerative processes, including Alzheimer""s disease, Parkinson""s disease and heart failure.
Programmed cell death serves as a major mechanism for the precise regulation of cell numbers and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes, and this form of programmed cell death has been termed apoptosis. Key elements of the apoptotic pathway include:
Death receptors: Apoptosis has been found to be induced via the stimulation of several different cell surface receptors in association with caspase activation. For example, the CD95 (APO-1, Fas) receptor ligand system is a critical mediator of several physiological and pathophysiological processes, including homeostasis of the peripheral lymphoid compartment and CTL-mediated target cell killing. Upon cross-linking by ligand or agonist antibody, the Fas receptor initiates a signal transduction cascade which leads to caspase-dependent programmed cell death.
Membrane alterations: In the early stages of apoptosis, changes occur at the cell surface and plasma membrane. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell.
Protease cascade: Signals leading to the activation of a family of intracellular cysteine proteases, the caspases, (Cysteinyl-aspartate-specific proteinases) play a pivotal role in the initiation and execution of apoptosis induced by various stimuli. At least 11 different members of caspases in mammalian cells have been identified. Among the best-characterized caspases is caspase-1 or ICE (Interleukin-1b-Converting Enzyme), which was originally identified as a cysteine protease responsible for processing of interleukin
Mitochondrial changes: Mitochondrial physiology is disrupted in cells undergoing either apoptosis or necrosis. During apoptosis mitochondrial permeability is altered and apoptosis specific protease activators are released from mitochondria. Specifically, the discontinuity of the outer mitochondrial membrane results in the redistribution of cytochrome C to the cytosol followed by subsequent depolarization of the inner mitochondrial membrane. Cytochrome C (Apaf-2) release further promotes caspase activation by binding to Apaf-1 and therefore activating Apaf-3 (caspase 9). AIF (apoptosis inducing factor), released in the cytoplasm, has proteolytic activity and is by itself sufficient to induce apoptosis.
DNA fragmentation: The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability (prelytic DNA fragmentation). In many systems, this DNA fragmentation has been shown to result from activation of an endogenous Ca2+ and Mg2+-dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA fragments.
Genetic studies in Caenorhabditis elegans had led to the identification of cell death genes (ced). The genes ced-3 and ced-4 are essential for cell death; ced-9 antagonizes the activities of ced-3 and ced-4, and thereby protects cells that should survive from any accidental activation of the death program. Caspases (cysteine aspartases) are the mammalian homologues of CED-3. CED-9 protein is homologous to a family of many members termed the Bcl-2 family (Bcl-2s) in reference to the first discovered mammalian cell death regulator. In both worm and mammalian cells, the anti-apoptotic members of the Bcl-2 family act upstream of the execution caspases somehow preventing their proteolytic processing into active killers.
Caspases appear to be present in most if not all cells in inactive proenzyme form, awaiting activation by cleavage. One of the killing mechanisms of cytotoxic T cells is a protease, granzyme B, that is delivered to the target cell by the T cell granules and triggers these latent proenzymes. There are endogenous triggers also, and the first to be discoveredxe2x80x94the C. elegans CED4 protein and its mammalian homologuexe2x80x94is particularly intriguing because of its mitochondrial origin. Thus CED4 could be the signal that initiates apoptosis under conditions of shutdown of cellular energy metabolism, or when there is a critical level of cell injury affecting mitochondrial respiration. In this way CED4 may act as the link between agents long known to be associated with mitochondrial injury, such as calcium and reactive oxygen species, and the initiation of apoptosis.
A second mitochondrial protein of enormous significance in apoptosis is BCL2, a mammalian homologue of the nematode CED9 protein. BCL2 has the tertiary structure of a bacterial pore-forming protein, and inserts into the outer membrane of mitochondria. Two main mechanisms of action have been proposed to connect Bcl-2s to caspases. In the first one, anti-apoptotic Bcl-2s would maintain cell survival by dragging caspases to intracellular membranes (probably the mitochondrial membrane) and by preventing their activation. The recently described mammalian protein Apaf-1 (apoptosis protease-activating factor 1) could be the mammalian equivalent of CED-4 and could be the physical link between Bcl-2s and caspases. In the second one, Bcl-2 would act by regulating the release from mitochondria of some caspases activators: cytochrome c and/or AIF (apoptosis-inducing factor). This crucial position of mitochondria in programmed cell death control is reinforced by the observation that mitochondria contribute to apoptosis signaling via the production of reactive oxygen species. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death.
There are other sources of death transducers, e.g., which activate the caspase cascade because of injury to or signals arising in other parts of the cell than mitochondria. For instance, the onco-suppressor protein p53 is activated following some types of DNA damage and can trigger apoptosis. One wayxe2x80x94but only one of severalxe2x80x94whereby this happens is through transcriptional activation of BAX7. The second messenger ceramide, a product of membrane-linked acid sphingomyelinase activation, may act as a signal for plasma membrane damage. And a powerful caspase-activating system is mediated by cytokine receptors of the tumor necrosis factor family, notably fas/apo1/CD95, TNF receptor I, and others. These receptors, on receiving a death stimulus from binding their ligand, initiate a series of protein-protein interactions, building a complex (the death initiating signaling complex or DISC) which eventually recruits and activates caspase.
Apoptosis plays an important role in the homeostasis and development of all tissues within an organism. In contrast to necrosis (cell death by accident), apoptosis is a well regulated physiological process. Any disturbance of the balance between cell proliferation and cell death maintained by apoptosis can result in serious disease, in particular cancer.
There is a need in the art for methods for the identification and analysis of compounds and biological factors which modulate apoptosis, such as those which can increase the rate of apoptosis, as well as compounds and biological factors which interfere with the induction of apoptosis, e.g., in Th cells.
Here, we report that TID1 encodes two mitochondrial matrix localized splice variants of 43 and 40 kDa, which we have named hTid-1L and hTid-1S, respectively. Both hTid-1L and hTid-1S retain their respective J domains and coimmunoprecipitate with mitochondrial Hsp70(mtHsp70). Expression of these proteins does not induce apoptosis, but surprisingly, expression of each of the two splice variants has opposing effects on a cell""s ability to respond to an exogenous apoptotic stimulus. hTid-1L expression increases apoptosis triggered by both tumor necrosis factor (TNF) and the DNA-damaging agent mitomycin c (MMC). A J domain mutant of hTid-1L is able to suppress apoptosis to levels well below control cells. In sharp contrast, hTid-1S is able to suppress apoptosis, and a J domain mutant of hTid-1S increases apoptosis. Expression of hTid-1L and hTid-1S affect cytochrome c release from the mitochondria and caspase 3 activation, both of which are downstream of the mitochondria in TNF signaling. However, hTid-1L and hTid-1S do not affect the rate of caspase 8 activation, which is upstream of the mitochondria. Hence, hTid-1L and hTid-1S are two mitochondrial matrix-localized proteins that can regulate apoptotic signal transduction and may comprise a mechanism by which the mitochondria amplify or dampen apoptotic signals.
We have found that mTid-1S, the murine homolog of the anti-apoptotic human TID1 encoded splice variant, hTid-1S, is specifically upregulated in Th2 cells upon activation induced with either anti-CD3xcex5 antibodies, or with PMA/ionomycin treatment. No upregulation is observed in Th1 cells upon activation. When a dominant negative mutant of hTid-1S is introduced into Th2 cells, these cells grow normally, but lose much of their resistance to AICD, and exhibit dramatically increased caspase 3 activity in response to anti-CD3xcex5 stimulation. Thus, activation-induced accumulation of hTid-1S contributes to resistance to AICD of Th2 cells.
Accordingly, in certain embodiment, the present invention specifically contemplates the use of agents which alter the ratio of Tid-1L to Tid-1S and/or selectively inhibit the activity of one of the splicing isoforms in order to sensitize or desensitize a cell to an apoptotic signal. For instance, compounds which inhibit the formation or activity of the Tid-1L form may be useful in desensitizing cells to apoptotic signals. Such agents may be useful in promoting the survival of tissue subject to degeneration, e.g., such agents may be protective against neurodegenerative disorders. Conversely, agents which selectively inhibit formation or activity of the Tid-1S form may be useful in sensitizing cells to apoptotic signals. Such agents may be useful in conjunction with chemotherapeutics or to enhance the body""s own ability to kill, e.g., virally infected cells or cancer cells.